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Carcinogenesis Advance Access published online on August 21, 2006
Carcinogenesis, doi:10.1093/carcin/bgl142
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
Received April 24, 2006
Revised July 28, 2006
Accepted July 28, 2006

MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION

Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients

Beatriz Zayas 1, Sara W. Stillwell 2, John S. Wishnok 2, Laura J. Trudel 2 *, Paul Skipper 2, Mimi C. Yu 3, Steven R. Tannenbaum 4, and Gerald N. Wogan 2

1 Universidad Metropolitana, San Juan, Puerto Rico; Biological Engineering Division, University of Minnesota, Minneapolis, MN 55455
2 Biological Engineering Division, University of Minnesota, Minneapolis, MN 55455
3 The Cancer Center, University of Minnesota, Minneapolis, MN 55455
4 Biological Engineering Division, University of Minnesota, Minneapolis, MN 55455; Chemistry Department, Massachusetts Institute of Technology, University of Minnesota, Minneapolis, MN 55455

* To whom correspondence should be addressed.
Laura J. Trudel, E-mail: ljtrudel{at}mit.edu


  Abstract

We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl adducts using an LC/MS/MS method with a 700 attomol (1 adduct in 109 bases) detection limit. Hemoglobin 4-ABP adduct levels were measured by GC/MS. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW = 443 Da) was added to each sample. Structural evidence and adduct quantification were determined by SRM, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444->328. The method was validated by analysis of DNA (100 µg each) from: calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 µg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5 to 80 dG-C8-ABP adducts per 109 bases; only 1/27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12 to 1960 picograms per gram hemoglobin. There was no correlation between levels of DNA and hemoglobin adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.


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